Evaluation of the pharmacokinetics of 2-carba-cyclic phosphatidic acid by liquid chromatography-triple quadrupole mass spectrometry.

Cyclic phosphatidic acid (cPA) is a lysophospholipid mediator that suppresses cancer metastasis and osteoarthritis. It also has neuroprotective roles in diseases such as multiple sclerosis and delayed neuronal death following transient ischemia. In order to take advantage of the properties of cPA for the development of new therapeutic strategies, we have synthesized several cPA derivatives and discovered 2-carba-cPA (2ccPA) as a promising candidate. To develop 2ccPA as a therapeutic agent, we investigated the pharmacokinetic profile of 2ccPA by liquid chromatography-triple quadrupole mass spectrometry in this study. When 2ccPA was administered intraperitoneally to mice at a dose of 1.6 mg/kg, the half-life of 2ccPA in plasma was 16 min. The 2ccPA, dosed intraperitoneally to mice at 16 mg/kg, distributed to each organ including brain at 20 min after dosing. It was found that 2ccPA was stable in neutral or alkaline conditions (e.g., intestine) but unstable in acidic conditions (e.g., stomach). When 2ccPA was orally administrated to rats as a gastro-resistant form using an enterosoluble capsule, plasma 2ccPA levels peaked at 2 h, slowly declined thereafter and persistently detected even at 10 h after administration. Here, we present the findings on the effect of the continuous release of 2ccPA from the capsule to reduce the lysophospholipase D activity and also decrease plasma levels of lysophosphatidic acid in rat. These findings will be useful in further studies for evaluating the application of 2ccPA in several disorders.

Effects of micro wet milling on bioaccessibility of phosphatidic acid and lysophosphatidic acid in komatsuna during in vitro digestion.

Foods rich in phosphatidic acid (PA) can ameliorate stomach ulcers in mice by hydrolysis of PA to lysophosphatidic acid (LPA). In this study, PA-rich komatsuna was produced using the micro wet milling (MWM) system, which can mill food products into micrometer-scale without causing detrimental factors such as frictional heat. To evaluate the efficiency of the MWM system in increasing PA and forming LPA, the availability of PA in the MWM komatsuna to hydrolyze into LPA under in vitro simulated gastrointestinal (GI) digestion conditions were investigated. The results showed that through effective MWM milling, komatsuna was sufficiently milled into smaller particles, and PA was abundantly produced in the milled komatsuna; the increased PA promoted LPA formation during digestion, resultant a dominant molecular species of 16:0 LPA which could effectively reduce ulcer lesions. These indicated that MWM can elevate the bioaccessibility of komatsuna PA and LPA in the GI tract, which will benefit the dietary treatment of stomach ulcers.

Phosphatidyl derivative of hydroxytyrosol. In vitro intestinal digestion, bioaccessibility, and its effect on antioxidant activity.

Intestinal digestion of phosphatidyl derivatives of HT (PHT) and its bioaccessibility under in vitro conditions was performed. First, an in vitro intestinal digestion model for phospholipids was developed. The impact of digestion in the antioxidant ability of PHT was also assayed. PHT was progressively hydrolyzed to lyso-PHT. However, digestion was slower than the phospholipid control. Nevertheless, most hydrolysis products were found at the micellar phase fraction, meaning a high bioaccessibility. Either PHT or digested PHT showed lower antioxidant activity than HT. However, PHT improved its antioxidant ability after digestion, likely related to lyso-PHT. As a summary, the synthetic phosphatidyl derivative of HT as PHT is recognized by phospholipases during simulation of intestinal digestion, although less efficiently than analogous phospholipids. Nevertheless, taking into account the bioaccessibility and the antioxidant activity of digested PHT, the potential of carriers of HT under the form of phospholipids might be of interest.

Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological activity toward autotaxin.

The efficient synthesis of 3-O-thia-cPAs (4a-d), sulfur analogues of cyclic phosphatidic acid (cPA), has been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic thiophosphate moiety. The present synthetic route enables the synthesis of 4a-d in only four steps from the commercially available glycidol. Preliminary biological experiments showed that 4a-d exhibited a similar inhibitory effect on autotaxin (ATX) as original cPA.

Synthesis and cytotoxic activity of two novel 1-dodecylthio-2-decyloxypropyl-3-phosphatidic acid conjugates with gemcitabine and cytosine arabinoside.

Cytosine arabinoside (ara-C) and gemcitabine (dFdC) are two standard chemotherapy drugs used in the treatment of patients with various cancers. To alter the pharmacokinetic and pharmacodynamic properties of these molecules, we conjugated a synthetic phospholipid to both ara-C and dFdC and investigated their chemotherapeutic potential. The dFdC conjugate had greater cytotoxic activity compared with the ara-C conjugate and demonstrated notable cytotoxicity against all human cell lines tested.

Anti-neovascular therapy by liposomal DPP-CNDAC targeted to angiogenic vessels.

We previously reported that liposomalized 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (DPP-CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, is quite useful for cancer therapy. On the other hand, for anti-neovascular therapy, we recently isolated peptides homing to angiogenic vessels from a phage-displayed random peptide library, and observed that peptide-modified liposomal adriamycin strongly suppressed tumor growth, perhaps through damaging angiogenic endothelial cells. In the present study, we modified DPP-CNDAC-liposomes with one of the angiogenic homing peptides, APRPG, and examined their antitumor activity. Three doses of APRPG-modified DPP-CNDAC-liposomes (15 mg/kg as CNDAC) strongly inhibited tumor growth compared with the same number of doses of unmodified DPP-CNDAC-liposomes. The life span was increased 31.8%, with one completely cured mouse out of the six mice treated. Since the accumulation of liposomes in the tumor tissue was not so much different between APRPG-liposomes and non-modified liposomes, the enhanced therapeutic efficacy may be explained as the alteration of targets, i.e. APRPG-modified DPP-CNDAC-liposomes caused tumor growth suppression through damage of angiogenic endothelial cells. Anti-neovascular therapy promises no drug resistance, and should be effective against essentially any kind of solid tumor; and thus the present results demonstrate another benefit of the therapy, namely, high efficacy of cancer treatment.

Co-existence of serum-dependent and serum-independent mechanisms for liposome clearance and involvement of non-Kupffer cells in liposome uptake by mouse liver.

The effect of serum on liver uptake of liposomes with different compositions was investigated using a single-pass liver perfusion technique. Among the liposomes tested are those containing CL, PA, DPGS, PE or glycolipids such as PI, GD, GT1b and aGM1. Liposomes containing PA, CL and DPGS showed high level of liver uptake in the absence of serum. Presence of serum decreased the total liver uptake for liposomes containing CL and PA by 50% and did not affect the level of liver uptake for DPGS-containing liposomes. The presence of serum, however, significantly increased the liposome uptake by the perfused liver for PG, PE and aGM1 liposomes. Liposomes containing PI showed a minimal liver uptake regardless of serum presence. Fluorescence microscopic studies using a dual fluorescence label system in combination with Kupffer cell elimination technique showed that, in addition to the dominant role of Kupffer cells in taking up liposomes, non-Kupffer cells may also be involved in taking up CL and DCP-containing liposomes. Competition experiments using various liposome compositions indicated that liposome uptake by the liver cells may involve different receptors. Serum activity in enhancing the liver uptake for PE- and aGM1-containing liposomes can be blocked by treatment of serum with EDTA, EGTA/Mg2+ and high temperature (56 degrees C), suggesting the involvement of complement system. Results from this study support the conclusion that blood clearance of liposomes by the liver involves two independent mechanisms, one requires serum opsonins and the other does not.

High-resolution fluorescent labeling of living cerebellar slices.

In the present study, we extend previous research on staining of living brain slices with fluorescent phospholipids. This new procedure allows high-resolution staining of specific cell types, in particular, Purkinje cells, in the cerebellar slice while not affecting the intrinsic electrical activity of the tissue. Four different nitrobenzoxadiole (NBD)-phospholipids were incorporated into living cerebellar slices via loading from small unilamellar vesicles (SUVs), composed of a carrier and the fluorescent lipid. The labeled acidic phospholipid, NBD-phosphatidic acid (NBD-PA), produced the highest resolution images with exquisite labeling of the dendritic fields. The label was incorporated predominantly into the Purkinje cell body (excluding the nucleus), with more diffuse staining in other cell types, including stellate, basket and granule cells. The labeled lipid concentration and composition of the carrier lipid were significant in determining the specificity of labeling. Labeling, which was optimal after a 1 h incubation, was present throughout the depth of the slice. This procedure provides a promising approach to fluorescent labeling that will allow simultaneous monitoring of changes in cellular morphology and electrophysiology of living brain slices.

Deacylation-reacylation cycle: a possible absorption mechanism for the novel lymphotropic antitumor agent dipalmitoylphosphatidylfluorouridine in rats.

Dipalmitoylphosphatidylfluorouridine (DPPF) is a potent antitumor agent that selectively gains access to the lymphatic system. To determine whether DPPF enters the lymph in an unmodified form, we administered DPPF orally to rats and analyzed lymph collected from a cannula in the thoracic duct. Although lymph was found to contain only very low levels of DPPF, two congeners of DPPF were detected at high levels. Instrumental analysis demonstrated that these congeners are 1-palmitoyl-2-arachidonoylphosphatidylfluorouridine (PAPF) and 1-palmitoyl-2-linoleoyl-phosphatidylfluorouridine (PLPF). PAPF and PLPF levels in thoracic lymph were shown to be approximately 30 times higher than those in plasma. These results suggest that DPPF is absorbed from the intestinal tract via the deacylation-reacylation cycle for the uptake of phospholipids and is selectively delivered to the lymphatic route after oral administration. DPPF is a candidate drug for the treatment of tumor metastasis, especially in cases where metastasis has occurred via the lymphatic route.

Role of cell cholesterol in modulating antineoplastic ether lipid uptake, membrane effects and cytotoxicity.

Membrane-interactive ether lipids (EL) exert toxic and antiproliferative effects on cancer cells in vitro. They appear to be selectively more toxic to cancer cells than to normal cells and thus they are ideal candidates for bone-marrow purging procedures. However, no conclusive explanation has yet been provided for this property. We now present some data indicating that the cholesterol concentration in the incubation medium modulates EL toxicity against the HL60 leukemic cell line in vitro. Furthermore, model membranes richer in cholesterol take up EL more slowly, and cell cholesterol enrichment of HL60 cells counteracts EL biophysical membrane interaction, but not toxicity, in our experimental model. However, the K562 cell line, a leukemia line less sensitive to EL toxic action, has higher levels of cell cholesterol. Our data provide evidence to explain differences in sensitivity to EL among different cell types and contribute to the understanding of the mechanism of action of EL.